Impedance-Based Cellular Assay to Measure Multiplicity of Infection of Influenza Virus

During influenza virus infection, the virus enters the cell through endocytosis and hijacks the host cell machinery, producing progeny virions. These get released, infecting neighboring cells, causing a cascade of infection.

To measure infectious influenza virus to cell ratio in culture — multiplicity of infection — add an appropriate density of freshly-obtained mammalian cell suspension into a microtiter plate.

The plate contains impedance-measuring gold microelectrodes embedded in the well bottom. Incubate, allowing cells to adhere to the well bottom and reach the exponential growth phase prior to infection.

Apply a small electrical potential between the microelectrodes. Cells adhered on the microelectrodes act as insulators, restricting current flow and increasing electrical resistance — impedance — which is measured.

Remove the media. Pipette the influenza virus suspension into the wells. Supplement with a suitable serine endoprotease.

During incubation, the serine endoprotease cleaves and activates the viral glycoprotein hemagglutinin, facilitating its binding to specific host cell receptors and subsequent virus cell entry.

Using host cell machinery, new viral particles are produced and released, infecting neighboring cells. As a result, the virus-infected cells undergo morphological changes and display cytopathic effects, leading to cell death — cells detach from the microelectrode surface, decreasing the impedance over time.

Determine the time taken for the impedance to reduce to half the initial impedance before virus addition. A shorter time frame suggests a higher multiplicity of viral infection.

To determine the correlation between the CIT₅₀ values and the multiplicity of infection, after culturing 3 x 10⁴ freshly split MDCK cells into each wall of the electronic microtiter plate for 24 hours, wash the cells two times with 100 microliters of fresh virus propagation medium per well per wash, and use a single channel pipette to add 100 microliters of viral suspension to each well.

When all of the virus dilutions have been added, gently load the plate into the cradle pocket of the instrument at 35 degrees Celsius, and begin monitoring the cell impedance every 15 minutes for at least 100 hours, as demonstrated.

After two cycles of measurements, click to "Pause" the apparatus and remove the E-plate from the cradle. Add 100 microliters of virus propagation medium supplemented with TPCK-trypsin to each well, and return the E-plate plate into the cradle pocket. Then, start the analysis.